TWI565715B - Blood coagulation factor vii and viia derivatives, conjugates and complexes comprising the same, and use thereof - Google Patents

Description

Derivatives of clotting factors VII and VIIa, conjugates and composites containing the same and uses thereof

The present invention relates to a Factor VII derivative, a Factor VIIa derivative, a FacVII and a FacVIIa conjugate (each prepared by attaching a polymer capable of prolonging blood half-life to the derivative), a FacVII and VIIa complex ( Each of the lines is prepared by linking a carrier to the conjugate, a gene encoding the derivative of FacVII and FacVIIa, a expression vector comprising the gene, and a transformed strain introduced into the expression vector, which is prepared using the transformed strain Method for preparing a derivative of FacVII and FacVIIa, a method for preparing a FacVIIa conjugate and a complex, and a FacVIIa complex prepared by the method comprising the derivative, the conjugate or the complex as an active ingredient for preventing or treating hemophilia A pharmaceutical composition for disease, and a pharmaceutical composition for promoting coagulation comprising the derivative, the conjugate or the complex as an active ingredient. Further, the present invention relates to a method for preventing or treating hemophilia or promoting coagulation comprising administering to a subject a therapeutically effective amount of the composition.

Currently, an estimated 140,000 people worldwide have hemophilia, showing a 20% annual increase. From a genetic point of view, one in every 10,000 people will have hemophilia, However, only about 25% of all patients are diagnosed or treated. Based on the pathogenesis, hemophilia is mainly divided into two types: one is hemophilia A due to the lack of factor VII (Factor VII, FacVII), and accounts for 80% of total hemophilia patients. He is hemophilia B due to the lack of factor XI (factor XI) and accounts for 20% of patients with total hemophilia. For the treatment of hemophilia, externally administered coagulation factors, but this treatment is problematic, 10 to 15% of all hemophilia A patients produce antibodies against coagulation factors, and in all hemophilia One to three percent of patients with disease B develop antibodies against coagulation factors.

On the other hand, FacVII is the main cause of hemophilia A, which accounts for more than half of hemophilia patients. The FacVII is mainly produced in the liver and is an enzyme composed of 406 amino acids, and includes glutamine at position 10. Acid gamma-carboxylation, aspartate N-glycation at positions 145 and 322, and O-glycation of serine at positions 52 and 60. Furthermore, FacVII has two epidermal growth factor (EGF-like) domains and one serine protease domain, and the single-chain FacVII is via arginine at position 152 and isoleucine at position 153. The cleavage is activated to produce a double-stranded FacVIIa composed of a light chain and a heavy chain. Since activated FacVIIa acts through a coagulation mechanism, unlike other coagulation factors, antibodies are not produced even when injected with high doses of FacVIIa. Therefore, it can be used for the treatment of patients with hemophilia A and patients having anti-FacVII antibodies due to conventional treatment, and is known as a method for solving the above problems.

However, although antibodies against FacVIIa are not produced, there are still problems in that high doses and frequent administration due to short half-lives in blood are required. Due to the short half-life, FacVIIa should be administered 2 to 3 times a day to treat hemophilia, and this frequent administration also causes serious obstacles to the prevention of hemophilia. In order to solve the problem of short half-life in the blood Many studies have suggested known micro-coating, liposome coating and various chemical modifications, but no successful results have been reported. In particular, attempts have been made to chemically modify the lysine residues or the N-terminus on the surface of FacVIIa to be chemically modified, or to carriers capable of prolonging the half-life of blood (eg, polyethylene glycol, albumin, transferrin, and immunoglobulin). Fragments), or cysteine residues, are inserted into regions that do not directly affect the activity of FacVIIa to facilitate binding to other carriers. However, the chemical modification of the lysine residue or the N-terminus on the surface of FacVIIa reduces the ability of FacVIIa to bind to platelet cell membranes. The carrier interferes with the activity of the enzyme when it is attached to other carriers. Insertion of a cysteine residue causes the formation of a non-specific disulfide bond, resulting in a decrease in enzyme activity. Therefore, many studies have been directed to the development of derivatives with improved half-life of blood without reducing the activity of FacVIIa, but no successful results have been reported.

rVIIa-FP (CSL Behring) prepared by fusing albumin to the C-terminus of FacVIIa was in the preclinical stage, and its blood half-life in rats increased to 6.7 times higher than that of native FacVIIa. However, it still has a very short half-life of 4.38 hours and is therefore not suitable for the treatment and prevention of hemophilia. The preparation of PEGLip-FVIIa (Omri) by the use of a PEGylated liposome formulation is also in the preclinical phase, but its blood half-life is only 2-fold higher than native FacVIIa.

Two products (MAXY-VII (Bayer/Maxygen) prepared by mutation of Gla domain and high saccharification of FacVIIa to have NN7128 (Novo/Neose) with extended blood half-life and 40K PEG saccharification to have The extended blood half-life is under clinical research, but its blood half-life is only five times higher than native FacVIIa. Therefore, they are not suitable for effective treatment and prevention of hemophilia.

Based on this background, the present inventors have been working to develop derivatives having improved blood half-life while retaining the maximum activity of FacVII and FacVIIa. As a result, it has been found that a derivative prepared by fusing a part of a superoxide dismutase (SOD1) sequence to the C-terminus of FacVII is easily compatible with a carrier which can prolong blood half-life (for example, polyethylene glycol, Albumin, transferrin and immunoglobulin fragments) bind without reducing FacVII or FacVIIa activity, in particular, specifically linking immunoglobulin Fc regions, non-peptidyl polymers, and FacVII or FacVIIa via covalent bond sites The present invention is accomplished by minimizing the reduction in activity and significantly increasing the blood half-life of the conjugate.

The object of the present invention is to provide a derivative of FacVII or an active form thereof, which has the amino acid sequence of Factor VII (Factor VII, FacVII) or its active form (Factor VIIa (Factor VIIa, FacVIIa)) and The peptide linker at the end.

A further object of the invention is to provide a polynucleotide encoding a derivative of FacVII or its active form of FacVIIa.

Still another object of the invention is to provide an expression vector comprising the polynucleotide.

Still another object of the present invention is to provide a transformant introduced into the expression vector.

Still another object of the present invention is to provide a method of using the transformant to prepare a derivative of the FacVII or its active form of FacVIIa.

Still another object of the present invention is to provide a conjugate of FacVII or an active form thereof, which is prepared by linking a polymer capable of extending blood half-life to a peptide linker of the derivative.

Still another object of the present invention is to provide a complex of FacVII or an active form thereof, FiVIIa, which is prepared by attaching a carrier capable of prolonging blood half-life to one end of the conjugate.

Still another object of the present invention is to provide a method for preparing the FacVIIa complex comprising the step of activating the FacVII complex.

Still another object of the present invention is to provide a FacVIIa composite prepared by the above method.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating hemophilia which comprises the derivative, conjugate or complex as an active ingredient.

Still another object of the present invention is to provide a pharmaceutical composition for coagulation comprising the derivative, conjugate or complex as an active ingredient.

Still another object of the present invention is to provide a method for preventing or treating hemophilia comprising the step of administering to a subject a therapeutically effective amount of the pharmaceutical composition for preventing or treating hemophilia.

Still another object of the present invention is to provide a method of promoting coagulation comprising the step of administering to a subject a therapeutically effective amount of the pharmaceutical composition for coagulation.

The derivative of FacVII or FacVIIa of the present invention can be combined with a carrier capable of improving blood half-life while maintaining the activity of FacVII or FacVIIa, and they can be widely used for the development of an effective preventive or therapeutic agent for hemophilia.

Figure 1a shows the appearance of FacVII-ATKAVC in the 293F cell line. Photograph of the results of the dot analysis; Figure 1b is a photograph showing the results of the Western blot analysis of the control group and the FacVII-GGGGSC in the 293F cell line; the 1c chart is the FacVII-ATKAVC showing the performance of the 293F cell line. And photographs of the results of Western blot analysis of the difference in molecular weight of FacVII-SOD1 1-149; Fig. 2 is a photograph showing the result of electrophoresis of purified FacVII-ATKAVC; and Fig. 3 is a photograph showing the FacVII-ATKAVC-PEG conjugate Electrophoresis Results Picture 4a is a photograph showing the results of Electrophoresis of FacVIIa-ATKAVC-PEG-Fc conjugate; Figure 4b is a photograph showing the results of Western dot analysis of the FacVIIa-ATKAVC-PEG-Fc conjugate; Figure 5 is a concentration dependent absorbance plot showing the in vitro activity of FacVII and FacVII-ATKAVC.

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In one aspect of achieving the above object, the present invention provides a derivative of FacVII or an active form thereof, FacVIIa, which has an amino acid sequence of Factor VII (Factor VII, FacVII) and a C thereof The peptide linker at the end.

As used herein, the term "Factor VII (Fac VII)", also known as proconvertin, is one of the factors involved in blood coagulation and has a size of 48 kDa, which is a gene with a size of 12.8 kb. It is encoded and produced primarily in the liver and is one of the vitamin K-dependent plasma proteins. It is known that FacVII binds to extravascular tissues (eg, serine protease precursors and smooth muscle cells, swollen Coagulation factor III on the surface of tumor tissue or activated leukocytes, thus activating coagulation factors IX and X, results in the initiation of extrinsic coagulation. In the present invention, FacVII may include native FacVIII, a chemically modified but retaining a normal FacVII derivative, and having at least 80% amino acid sequence homology with native FacVII (preferably 85%, 90% or 95). % amino acid sequence homology, more preferably 98% or 99% amino acid sequence homology) but at the same time they retain a variant of the normal activity of native FacVII. However, the sequence homology is not limited thereto as long as they show the activity of the native FacVII.

The term "factor VIIa (Factor VIIa, FacVIIa)" as used herein means the active form of Factor VII (Factor VII, FacVII), the single-chain FacVII line via arginine at position 152 and at position 153 The shear between isoleucine is activated to produce a double-stranded FacVIIa composed of a light chain and a heavy chain. Since activated FacVIIa acts through a coagulation mechanism, unlike other coagulation factors, antibodies are not produced even when injected with high doses of FacVIIa. In the present invention, FacVII may include native FacVII, a chemically modified but retaining the normal activity of natural FacVII, having at least 80% amino acid sequence homology with native FacVII (preferably 85%, 90% or 95). % amino acid sequence homology, more preferably 98% or 99% amino acid sequence homology) but at the same time they retain a variant of the normal activity of native FacVII. However, the sequence homology is not limited thereto as long as they show the activity of the native FacVII.

The term "linker" as used herein basically means that two different fusions can be linked using hydrogen bonding, electrostatic interaction, van der Waals force, disulfide bond, salt bridge, hydrophobic interaction, covalent bond, and the like. Tools for partners (eg biopolymers). Preferably, it may have at least one cysteine involved in at least one disulfide bond under physiological conditions or other standard peptide conditions (eg, peptide purification conditions, peptide storage conditions). It can use the cysteine as a reactive group to link the fusion partner And the disulfide bond. In addition, the linker serves as a hub for providing a predetermined space between the carriers, or as a hub for providing the elasticity or rigidity of the fusion protein, and simply as a link to each fusion partner. In the present invention, the linker is, but not particularly limited to, a C-terminus linked to the C-terminus of FacVII or FacVIIa to link a carrier capable of prolonging the half-life of the blood, and preferably a C-terminus of the peptide linker Cysteine residue. Preferably, it may be a partial sequence of SOD1 (superoxide dismutase 1) (SEQ ID NO. 30), and more preferably a partial sequence of 1 to 149 (SEQ ID NQ. 31) selected from the sequence of SOD1, More preferably, from 1 to 90 (SEQ ID NO. 32) of the SOD1 sequence, and even more preferably, from 1 to 25 (SEQ ID NO. 33) of the SOD1 sequence, the best, from the 1 of the SOD1 sequence to 6 (SEQ ID NO. 5).

The term "SOD1 (superoxide dismutase 1)" as used herein means the catalytic disproportionation of reactive oxygen, peroxide ions to oxygen and hydrogen peroxide, which is known to be expressed in all cells exposed to An important antioxidant defense in oxygen. In the present invention, the SOD1 is used as a peptide linker capable of linking FacVII and capable of prolonging the blood half-life carrier. SOD1, which is commonly found in the body, is used as a linker to reduce the immunogenicity of the linker. VLKG (proline-leucine-lysine-glycine) in the sequence of the peptide linker SOD1 can be recognized by the FacVIIa derivative and sheared by the self-shear point sequence IPRI (different Substituted by leucine-valine-arginine-isoleucine. Due to the substitution of this self-shearing sequence, the linker region that is not required for activation upon activation can be removed by the FacVIIa derivative.

In the present invention, the self-shearing point is a point comprising a specific sequence in which the multi-peptide has a corresponding specific sequence in its sequence and is recognized and cleaved.

The term "FacVII derivative" as used herein means modified The FacVII, the modified FacVII line is composed of an amino acid sequence prepared by linking the peptide linker to the C-terminus of FacVII. The FacVII derivative of the present invention means a form before activation and is changed to a FacVIIa derivative upon activation via a specific method. In the present invention, the FacVII derivative and the FacVIIa derivative may have the same meaning except for the preparation process of a conjugate or the like in a specific step, for example. In the present invention, the FacVII derivative is, but not particularly limited to, a multi-peptide prepared by linking the ATKVC (SEQ ID NO. 5) of the SOD1 sequence to the C-terminus of the FacVII derivative. (SEQ ID NO. 9), a multi-peptide (SEQ ID NO. 13) prepared by linking GGGGSC (SEQ ID NO. 10) to the C-terminus of the FacVII derivative, by ligation of the SOD1 sequence from 1 a polypeptide (SEQ ID NO. 14) prepared by the amino acid sequence of 149 to the C-terminus of the FacVII derivative, by the amino acid sequence from 1 to 90 linked to the SOD1 sequence to the FacVII derivative Polypeptide (SEQ ID NO. 34) prepared at the C-terminus, a multi-peptide prepared by linking the amino acid sequence of 1 to 25 of the SOD1 sequence to the C-terminus of the FacVII derivative (SEQ ID NO.35), by ligating the polypeptide (SEQ ID NO. 20) prepared by linking the amino acid sequence of 1 to 149 of the mutated SOD1 sequence to the C-terminus of the FacVII derivative a multi-peptide (SEQ ID NO. 27) prepared from the amino acid sequence of the mutated SOD1 sequence from 1 to 90 to the C-terminus of the FacVII derivative, by linking the mutated SOD1 sequence from 1 to 25 amino acid sequence to the derivative of FacVII Polypeptide (SEQ ID NO. 24) prepared at the C-terminus of the compound.

The term "FacVIIa derivative" as used herein means the active form of the FacVII derivative which has the same amino acid sequence as the FacVII derivative but which is cleaved at positions 152 and 153. It is activated. In the present invention, the FacVIIa derivative is, but not particularly limited to, by linking the SOD1 a multi-peptide (SEQ ID NO. 9) prepared from the ATKAVC (SEQ ID NO. 5) of the sequence from 1 to 6 to the C-terminus of the FacVIIa derivative, by linking GGGGSC (SEQ ID NO. 10) to the Polypeptide (SEQ ID NO. 13) prepared at the C-terminus of the FacVIIa derivative, a multi-peptide prepared by linking the amino acid sequence from 1 to 149 of the SOD1 sequence to the C-terminus of the FacVIIa derivative (SEQ ID NO. 14), by ligating the polypeptide (SEQ ID NO. 34) prepared by linking the amino acid sequence of 1 to 90 of the SOD1 sequence to the C-terminus of the FacVIIa derivative a multi-peptide (SEQ ID NO. 35) prepared from the amino acid sequence of 1 to 25 of the SOD1 sequence to the C-terminus of the FacVIIa derivative, an amine from 1 to 149 linked to the mutated SOD1 sequence a peptide derived from the C-terminus of the FacVIIa derivative (SEQ ID NO. 20) by linking the amino acid sequence from 1 to 90 of the mutated SOD1 sequence to the FacVIIa derivative a multi-peptide prepared at the C-terminus (SEQ ID NO. 27), or a multi-peptide prepared by linking the amino acid sequence of 1 to 25 of the mutated SOD1 sequence to the C-terminus of the FacVIIa derivative (SEQ ID NO. 24).

The inventors studied the characteristics of activated FacVII and intend to develop derivatives having improved blood half-life without reducing the activity of FacVIIa. The unactivated FacVII line is a single-chain FacVII that links the light and heavy chains and exposes only the N-terminus of the light chain. However, when FacVII is changed to FacVIIa, the active site of the heavy chain is exposed by shearing between arginine at position 152 and isoleucine at position 153, and the isoleucine at the exposed position 153 becomes The N-terminus of the heavy chain. The N-terminus of the heavy and light chains plays an important role in the activation of FacVIIa, so the N-terminal junction may reduce the activity of FacVII compared to native FacVII.

To this end, the present invention provides a method for preparing a FacVII derivative by using the SOD1 peptide sequence fragment as a linker, the peptide fragment comprising cysteine The structure does not expose to the outside and therefore does not participate in the disulfide bond. Furthermore, a self-shearing point sequence which can be recognized and cleaved via a FacVIIa derivative is inserted as a peptide fragment for ligation of a linker, and thus a linker which is not required for activation can be removed. The present invention provides a FacVII derivative having a cysteine fragment containing a free peptide at the C-terminus. As a result, a dimeric form of the FacVII derivative was found. As a result, it was found that a dimer type of the FacVII derivative was produced at the lowest level during the culture, and the FacVII derivative can easily form a conjugate with a carrier capable of prolonging the blood half-life, thereby compensating for the natural FacVII and by simple Disadvantages of derivatives prepared by the insertion of cysteine into FacVIIa-.

Therefore, the preparation of the conjugate is carried out by attaching the C-terminus of the FacVII or FacVIIa derivative of the present invention to a substance capable of remarkably improving blood half-life, maintaining coagulation function, and significantly increasing medication compliance, thereby preparing compared to known The product has a product that improves coagulation and prevents or treats hemophilia.

In another aspect, the present invention provides a polynucleotide encoding the FacVII derivative, a expression vector comprising the polynucleotide, a transformant introduced into the expression vector to express the FacVII derivative, and using the transformant to prepare Method of the FacVII derivative.

The polynucleotide encoding the FacVII derivative provided by the present invention is, but not particularly limited to, a polynucleotide prepared by linking a region encoding a FacVII region to a region encoding a peptide, preferably encoded by linking the polynucleotide. Polynucleotide (SEQ ID NO. 8) of the SOD1 sequence of ATKAVC (SEQ ID NO. 5) from 1 to 6 to the C-terminus of the FacVII derivative, encoded by the polynucleotide (SEQ ID NO. 8) A polynucleotide (SEQ ID NO. 12) of the polypeptide (SEQ ID NO. 13) prepared by linking GGGGSC (SEQ ID NO. 10) to the C-terminus of the FacVII derivative, encoded by linking Polynucleotide (SEQ ID NO. 15) of the polypeptide (SEQ ID NO. 14) prepared from the amino acid sequence of 1 to 149 of the SOD1 sequence to the C-terminus of the FacVII derivative, encoded by linking Polynucleotide (SEQ ID NO. 21) of the polypeptide (SEQ ID NO. 20) prepared from the amino acid sequence of 1 to 149 of the mutated SOD1 sequence to the C-terminus of the FacVII derivative, encoding Polynucleotide (SEQ ID NO. 28) of Polypeptide (SEQ ID NO. 27) prepared by linking the amino acid sequence from 1 to 90 of the mutated SOD1 sequence to the C-terminus of the FacVII derivative (SEQ ID NO. Or a polynucleotide encoding the polypeptide (SEQ ID NO. 24) prepared by linking the amino acid sequence of 1 to 25 of the mutated SOD1 sequence to the C-terminus of the FacVII derivative (SEQ ID NO. 24) NO.25).

The expression vector comprising the polynucleotide encoding the FacVII derivative provided by the present invention is, but not particularly limited to, a vector capable of replicating and/or expressing the polynucleotide in a eukaryotic or prokaryotic cell, the cell comprising a mammal Cells (eg, humans, monkeys, rabbits, rats, hamsters, mouse cells, etc.), plant cells, yeast cells, insect cells, or bacterial cells (eg, E. coli, etc.), and are preferably operatively linked To a suitable promoter to express the polynucleotide in a host cell, and to comprise at least one selectable marker. More preferably, the expression vector can be prepared by introducing the polynucleotide into a phage, plastid, mucin, minichromosome, viral vector or retroviral vector. Most preferably, the polynucleotide comprising a FacVII derivative prepared by ligating a polynucleotide encoding ATKAVC (SEQ ID NO. 5) from 1 to 6 encoding the SOD1 sequence to the 3' end of the FVII gene. Expression vector pX0GC-FVII-ATKAVC, comprising the expression vector pX0GC- of the polynucleotide encoding the FacVII derivative prepared by ligating the polynucleotide encoding GGGGSC (SEQ ID NO. 10) to the 3' end of the FVII gene. FVII-GGGGSC, comprising a polynucleotide to the FVII gene by ligating the amino acid sequence from 1 to 149 (SEQ ID NO. 14) encoding the SOD1 sequence Expression vector pX0GC-FVII-SOD1 1-149 of the polynucleotide encoding the FacVII derivative prepared at the 3' end, comprising a polynucleoside by linking an amino acid from 1 to 149 encoding the mutated SOD1 sequence The expression vector pX0GC-FVII-SOD1 IPRI of the polynucleotide (SEQ ID NO. 21) encoding the FacVII derivative prepared by acid to the 3' end of the FacVII gene, including self-ligation encoding the mutated SOD1 sequence by ligation The expression vector pX0GC-FVII-SOD1 1-90 IPRI of the polynucleotide encoding the FacVII derivative (SEQ ID NO. 28) prepared by the polynucleotide of amino acid of 90 to the 3' end of the FVII gene, or Including the polynucleotide encoding the FacVII derivative (SEQ ID NO. 25) prepared by ligating a polynucleotide encoding an amino acid of 1 to 25 of the mutated SOD1 sequence to the 3' end of the FVII gene. Expression vector pX0GC-FVII-SOD1 1-25 IPRI.

The transforming strain introduced into the expression vector provided by the present invention is, but not particularly limited to, a bacterial cell transformed by introducing the expression vector (for example, E. coli, Streptomyces, and Salmonella) Salmonella typhimurium); yeast cells (eg, Pichia pastoris); insect cells (eg, Drosophila and Spodoptera Sf9); animal cells (eg, CHO) , COS, NSO, 293 and human melanoma cells; or plant cells. Preferably, HMF709 prepared by introducing the expression vector into a 293F or CHO cell strain, and more preferably by introducing the expression vector pX0GC-FVII-ATKAVC to a CHO cell line.

The method for producing the FacVII derivative provided by the present invention comprises the steps of (i) cultivating the transformant to obtain a culture solution, and (ii) recovering the FacVII derivative from the culture solution.

The method further comprises the step of activating the recovered FacVII derivative, The FacVIIa derivative was prepared from the prepared FacVII derivative. This activation method is the same as the above method.

The present inventors prepared a polynucleotide comprising the FacVII derivative prepared by ligating a polynucleotide encoding ATKAVC (SEQ ID NO. 5) from 1 to 6 encoding the SOD1 sequence to the 3' end of the FVII gene. The expression vector pX0GC-FVII-ATKAVC (Example 2-1) was introduced into the 293F cell line (Example 3-1) or the CHO cell line (Example 3-2) to obtain a transformant. . Subsequently, the FacVII derivative was expressed from the transformant, and the expressed FacVII derivative was purified (Example 4, Figure 2). The represented FacVII derivative was activated to prepare the FacVIIa derivative, followed by comparing the activity of the FacVIIa derivative with native FacVIIa (Example 6, Figure 4). As a result, it was found that the FacVIIa derivative prepared from the FacVII derivative of the present invention showed activity equivalent to that of natural FacVIIa. Therefore, the plant line showing the highest expression level of the FacVII derivative was selected from the transformant strain prepared by introducing the expression vector pX0GC-FVII-ATKAVC to CHO cells, and was designated as "HMF709", and was deposited in Korea. A typical strain preservation center, Korean Institute for Bioscience and Biotechnology (Korean Collection for Bioscience and Biotechnology (111 Gwahangno, Yuseong-gu, Daejeon, Korea)), the registration number is "KCTC12022BP".

In still another aspect, the present invention provides a conjugate of FacVII or an active form thereof, the conjugate, which is prepared by linking a polymer capable of prolonging blood half-life to a peptide linker of the FacVII derivative.

The polymer of the present invention may be a polymer (e.g., polyethylene glycol) capable of prolonging blood half-life, and is selected from a protein carrier (e.g., immunoglobulin fragment, transferrin, antibody, and albumin).

The present invention provides a conjugate prepared by using a non-peptidyl polymer as a linker in vitro to link the FacVII derivative with the protein carrier without using a genetic recombination method.

The non-peptidyl polymer system of the present invention means a non-peptidyl polymer designed to resist degradation by a plurality of enzymes or immune molecules in blood or serum, and the non-peptidyl polymer is not limited to the following, optional Self-polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid Polymer, chitin, hyaluronic acid, and combinations thereof. And the non-peptidyl polymer can be linked to each other via any kind of covalent bond other than the peptide bond. Further, derivatives known in the art and derivatives thereof which can be readily prepared by techniques known in the art are within the scope of the invention. In the present invention, the non-peptidyl polymer may be linked to the FacVII derivative or the linker of the FacVIIa derivative. Non-peptidyl polymers can be linked to various attachment sites of the peptide linker. Preferably, the non-peptidyl polymer can be linked to the C-terminus of the peptide synthon of the FacVII derivative or the FacVIIa derivative.

The non-peptidyl polymer may comprise a reactive group, which may include, but is not limited to, aldehyde group, propionaldehyde, butyraldehyde, maleimide or amber imine (amber a propionate) Imino ester, carboxymethyl amber succinimide, hydroxy amber succinimide or amber succinimide carbonate). Further, the non-peptidyl polymer may have a single reactive group or a double reactive group. If the non-peptidyl polymer comprises two or more reactive groups, it may be linked to the linker of the FacVII derivative by one reactive group, and also linked to other carriers by other reactive groups (eg, antibody, immunoglobulin) Protein fragment, albumin or transferrin). For example, when the non-peptide group has a reactive acetaldehyde group at one end and maleic imine, ortho-pyridyl disulfide at the other end In the case of a thiol-reactive group, the non-specific reaction can be minimized and it is effective for selective binding of the FacVII derivative or the FacVIIa derivative to the carrier at both ends of the non-peptidyl polymer. The final product resulting from the reductive alkylation reaction of the acetaldehyde bond may be more stable than the guanamine bond, and further, the acetaldehyde reactive group selectively reacts with the amine terminal of the carrier at a low pH, and A covalent bond can be formed with a lysine residue at a high pH (eg, pH 9.0).

In still another aspect, the present invention provides a complex of FacVII or an active form thereof, which is prepared by linking the derivative of the FacVII or its active form of FacVIIa with the immunoglobulin Fc region with the non-peptidyl polymer.

The FacVII complex linked to a carrier (eg, antibody, immunoglobulin fragment, albumin, and transferrin), in particular, the immunoglobulin Fc linked via the non-peptidyl polymer can be prepared by the following steps: (1) covalently linking a non-peptidyl polymer having an aldehyde or amber quinone imine derivative reactive group at one end to an amine group of the immunoglobulin Fc; recovering from the reaction mixture of the step (1) includes an amine group and a conjugate of an immunoglobulin Fc region covalently linked to the non-peptidyl polymer; (3) covalently linking the FacVII derivative to the recovered conjugate having a pentamidine, an ortho-pyridyl disulfide or a thiol The other end of the non-peptidyl polymer of the reactive group is used to produce a FacVII complex having the immunoglobulin Fc region and the FacVII derivative at each end of the non-peptidyl polymer; and (4) activation in step (3) The resulting FacVII conjugate is used to generate a FacVIIa complex that links FacVIIa and the immunoglobulin Fc region via the non-peptidyl polymer.

Furthermore, the FacVII complex can be prepared by the following steps: (1) covalently linking a non-peptidyl polymer having a maleimine, an ortho-pyridyl disulfide or a thiol-reactive group at one end to the C-terminal thiol group of FacVII derivative; from step (1) The reaction mixture recovers a conjugate comprising a FacVII derivative covalently linked to the non-peptidyl polymer; (3) covalently linking the immunoglobulin Fc region to the recovered conjugate having an aldehyde or amber imine derivative The other end of the non-peptidyl polymer of the reactive group is used to produce a FacVII complex having the immunoglobulin Fc region and the FacVII derivative at each end of the non-peptidyl polymer; and (4) activation in the step ( 3) The FaVII conjugate produced to produce a FacVIIa complex having FacVIIa linked via the non-peptidyl polymer and the immunoglobulin Fc region.

Furthermore, the FacVIIa complex can be prepared by the following steps: (1) covalently linking a non-peptidyl polymer having a maleimine, an ortho-pyridyl disulfide or a thiol-reactive group at one end to the a C-terminal thiol group of the FacVIIa derivative; recovering a conjugate comprising the FacVII derivative covalently linked to the non-peptidyl polymer from the reaction mixture of the step (1); and (3) covalently linking the immunoglobulin Fc The other end of the non-peptidyl polymer having an aldehyde or amber quinone imine derivative reactive group in the recovered conjugate to produce the immunoglobulin Fc region at each end of the non-peptidyl polymer and The FacVIIa complex of the FacVIIa derivative.

In another aspect, the non-peptidyl polymer can include two or three reactive ends, which can be the same or different from each other. For example, it may have a maleimine group at one end and an acetaldehyde group, a propionaldehyde group or a butyraldehyde group at the other end. When poly(ethylene glycol) has a hydroxyl reactive group at both ends thereof for use as the non-peptidyl polymer, the hydroxyl group may be activated to various reactive groups via a known chemical reaction, or may be commercially available. The modified reactive group of poly(ethylene glycol) can be used to prepare the FacVII conjugates and complexes of the present invention.

Therefore, the non-peptidyl polymer included in the FacVII conjugate and composite of the present invention preferably has a methyl group at one end and a male 醯 at the other end. a non-peptidyl polymer of an imine, an ortho-pyridyl disulfide or a thiol-reactive group, and more preferably a maleimide, an ortho-pyridyl disulfide or a thiol-reactive group at one end and A non-peptidyl polymer having an aldehyde or amber quinone imine derivative reactive group at the other end, and more preferably a non-peptidyl polymer having a maleic imine reactive group and an aldehyde group at both ends.

The FacVII derivative used to prepare the conjugate or complex of the present invention may be an inactive or activated FacVIIa derivative. However, in order to prevent degradation due to activated FacVIIa during the preparation of the conjugate using the FacVIIa derivative, it is preferred to use the FacVII system.

As a vehicle, the Fc region can be obtained from natural forms isolated from humans and other animals including cattle, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs. Furthermore, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, and IgM, or a combination thereof or a mixture thereof. Preferably, it is derived from IgG or IgM (the most abundant protein in human blood) and is optimally derived from IgG (known to enhance the half-life of the ligand-binding protein). The immunoglobulin Fc region can be obtained from the native immunoglobulin by isolating the intact immunoglobulin from a human or animal organism and treating it with a specific protease enzyme, and can also be obtained by autotransformation of cells by recombinant techniques. Preferably, it is a recombinant human immunoglobulin Fc region from E. coli. On the other hand, IgG is classified into IgG1, IgG2, IgG3, and IgG4 subclasses, and the present invention includes combinations thereof and hybridization. Preferred are IgG2 and IgG4 subclasses, and the preferred ones are Fc regions of IgG4 that have little effector function (eg, complement dependent cytotoxicity (CDC).

That is, as the drug carrier of the present invention, the optimal immunoglobulin The Fc region is a human IgG4-derived non-glycosylated Fc region. The human-derived Fc region is better than the non-human-derived Fc region, which is used as an antigen in humans and causes an unwanted immune response (eg, new antibody production against the antigen).

A peptide linker for a fusion protein obtained by a conventional inframe fusion method has a drawback of being easily cleaved by a proteolytic enzyme in the body, and thus it is not possible to obtain an active drug by a carrier as expected. The full efficacy of serum half-life. However, in the present invention, a polymer having an antiproteolytic enzyme can be used to maintain the serum half-life of the peptide similar to the carrier. Therefore, any non-peptidyl polymer, and a polymer having the aforementioned function, that is, a polymer having an antiproteolytic enzyme in vivo, can be used without limitation. The non-peptidyl polymer has a molecular weight in the range of 1 to 100 kDa, preferably 1 to 40 kDa. The non-peptidyl polymer of the present invention is linked to the immunoglobulin Fc region and may be a polymer or a combination of different kinds of polymers.

In a specific embodiment of the invention, the in vitro activity of the FacVII conjugate is determined. The present invention is intended to minimize the reduction in activity due to site-specific ligation of FacVII and the non-peptidyl polymer. Therefore, the FacVII-ATKAVC and FacVII-ATKAVC-40kDa PEG activities were determined using the natural FacVII and FacVII-40 kDa PEG as a control group (Example 7). The in vitro activity of the N-terminally PEGylated FacVII-40 kDa PEG was found to be approximately 11% compared to that of the FacVII, and the C-terminally PEGylated FacVII-ATKAVC-40 kDa PEG was in vitro compared to the FacVII-ATKAVC. The number of people seen is about 29%. That is, the C-terminus of the PEG FacⅦ-ATKAVC-40 kDa PEG maintain the activity about 2.5 times higher than the N-terminus of the PEG of the FacⅦ-40 kDa PEG EC _50, it may be represented by a specific point of engagement in use ATKAVC Higher levels maintained this FacVII activity (Table 2).

In another embodiment, the in vitro activity of the complex prepared by linking a non-peptidyl polymer and an immunoglobulin Fc region to a FacVII conjugate is determined (Example 8). As a result, it was found that the in vitro activity of FacVIIa-ATKAVC-PEG-Fc was about 45% compared with that of FacVIIa-ATKAVC (Table 3), indicating that the complex linked to the non-peptidyl polymer and the immunoglobulin Fc region can improve blood. The half-life carrier and the simultaneous maintenance of the FacVII activity are widely used to develop more effective prophylactic or therapeutic agents for hemophilia.

In still another aspect, the invention provides a method for preparing a FacVIIa conjugate comprising the step of activating the FacVII conjugate, and the invention provides a FacVIIa conjugate prepared by the method. In detail, the method for preparing the FacVIIa conjugate may comprise the steps of: (i) covalently linking a non-peptidyl polymer capable of prolonging blood half-life to a C-terminal thiol group of the FacVII derivative; (ii) recovering from the non- A peptidyl polymer is attached to the FacVII conjugate of the FacVII derivative; and (iii) the recovered FacVII conjugate is activated to produce a FacVIIa conjugate having the peptide polymer attached to the FacVIIa region.

Further, the method comprises the steps of: (i) covalently linking the non-peptidyl polymer capable of prolonging the half-life of blood to the thiol group at the C-terminus of the FacVIIa derivative; and (ii) recovering the linkage to the FacVIIa by the non-peptidyl polymer The derivative constitutes the FacVIIa conjugate for producing a FacVIIa conjugate having the non-peptidyl polymer attached to the FacVIIa region.

The non-peptidyl polymer system used in the method to increase blood half-life is the same as described above, and the method for activating the FacVII or FacVII conjugate is, but not limited to, by attaching it to an anion exchange column. Activate the FacVII or On-column activation (auto-activation) of a FacVII conjugate, or activation in a solution that activates the FacVII or FacVII conjugate by reacting it in a solution phase (in-solution) Activation). In particular, the activation in the column is also referred to as solid-phase activation, and by the addition of the FacVII or FacVII conjugate to the anion exchange column, no additional components are required for "auto-activation". get on. Conversely, the activation system in this solution is a method of inducing activation of FacVII, taking into account various factors required for activation of FacVII (eg, calcium ion concentration, pH, temperature, and FacvII concentration).

The present inventors have confirmed that the half-life of the conjugate prepared by linking the immunoglobulin fragment to the native FacVII via the non-peptidyl linker is increased by about half compared to the native FacVII having no immunoglobulin fragment (Korean Patent Publication No. 2010-0062860). 200 times. It is known that this increased half-life is not due to FacVII, but is due to the non-peptidyl linker and the immunoglobulin fragment. Therefore, the conjugate prepared by using the prepared FacVII derivative is also expected to have an increased half life.

In still another aspect, the present invention provides a pharmaceutical composition for preventing or treating hemophilia or a pharmaceutical composition for coagulation comprising a derivative of FacVII or an active form of FacVIIa, a conjugate of FacVII or its active form FacVIIa A complex of FacVII or its active form of FacVIIa is used as an active ingredient.

Furthermore, the invention provides a method for preventing or treating hemophilia or for promoting coagulation comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition.

The term "prevention" as used herein means all effects of inhibiting or preventing the onset of hemophilia by simultaneous administration of a composition of the invention, and the term "treatment" means simultaneously by administering a composition of the invention. Improve or beneficially alter all effects of hemophilia symptoms that have occurred.

In the present invention, the method for promoting coagulation is to promote a blood coagulation by preparing a derivative, a conjugate or a complex which has an apparently increased blood half-life from a blood coagulation factor FacVII having a short half-life or its active form FacVIIa. .

Further, the pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein means a carrier or diluent that does not cause significant irritation to the organism and does not abrogate the biological activity and properties of the administered compound. For oral administration, pharmaceutically acceptable carriers may include binding agents, lubricants, disintegrating agents, excipients, solubilizing agents, dispersing agents, stabilizers, suspending agents, coloring agents, and flavoring agents. For use in the injection, a pharmaceutically acceptable carrier can include a buffer, a preservative, an analgesic, a solubilizing agent, an isotonicity agent, and a stabilizer. For topical administration, a pharmaceutically acceptable carrier can include a base, excipient, lubricant, and preservative. The pharmaceutical composition of the present invention can be combined with the above pharmaceutically acceptable carrier to be formulated into various dosage forms. For example, when administered orally, the pharmaceutical composition can be formulated into a tablet, an ingot, a capsule, an elixir, a suspension, a syrup or a wafer. When used in an injection, the pharmaceutical composition can be formulated into a unit dosage form, such as a multi-dose container or a ampule in a single dosage form. The pharmaceutical composition can also be formulated into solutions, suspensions, lozenges, pills, capsules and long-acting preparations.

In another aspect, examples of carriers, excipients, and diluents suitable for use in pharmaceutical formulations include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch , acacia, alginate, gelatin, calcium phosphate, calcium citrate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate , talc, magnesium stearate and mineral oil. In addition, the pharmaceutical preparation may further comprise a filler, an anti-caking agent, Lubricants, humectants, flavoring agents and antibacterial agents.

In still another aspect, the present invention provides a method for treating hemophilia comprising administering to a subject having hemophilia a therapeutically effective amount comprising the derivative, conjugate or complex as an active ingredient for prevention or A pharmaceutical composition for the treatment of hemophilia. In this regard, the pharmaceutical composition can be administered alone or simultaneously or sequentially with other therapeutic agents.

As used herein, the term "administering" means introducing a predetermined amount of a substance into a patient by some suitable method. The composition can be administered by any conventional route as long as it can reach the desired tissue. Various administration modes may be employed, including intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, and rectal, but the invention is not limited to these examples. Investment mode. However, since the peptide is digested upon oral administration, the active ingredient in the composition for oral administration should be coated or formulated to protect against degradation in the stomach. Preferably, the multimer can be administered in an injectable form. In addition, the pharmaceutical composition can be administered using certain devices that deliver the active ingredient to the target cells.

Furthermore, the pharmaceutical composition of the present invention can be determined according to various factors, including the type of the disease to be treated, the route of administration, the age, weight and severity of the disease, and the type of drug used as the active ingredient.

The pharmaceutical composition of the present invention exhibits excellent sustained efficacy and strength in vivo, and thus significantly reduces the frequency and frequency of administration for preventing or treating hemophilia or for promoting coagulation.

[Embodiment]

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the examples are for illustrative purposes only and the invention is not intended to be limited by the examples.

Example 1: Preparation of Expression Vector Containing FacVII Gene

First, a polymerase chain reaction (PCR) technique was used to obtain a human factor VII gene containing a message sequence. In order to amplify the Factor VII (FacVII) gene, a human fetal liver cDNA library (TAKARA BIO USA) was used as a template, and the forward and reverse primers of the following SEQ ID NOS. 1 and 2 were used. PCR (denaturation at 95 ° C for 1 minute; 30 cycles (95 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 90 seconds) at 68 ° C for 5 minutes). At this time, in order to facilitate colonization, the recognition region of the restriction enzyme BamHI was inserted into the primer of SEQ ID NO. 1, and the recognition region in which the restriction enzyme XhoI was inserted in the primer of SEQ ID NO. Subsequently, the nucleotide sequence of the approximately 1.3 kb PCR product obtained by PCR (SEQ ID NOS. 3 and 4) was examined.

VII BHISS F: 5'-cccggatccatggtctcccaggccctcaggctcc-3' (SEQ ID NO. 1)

VII XhoIAS R: 5'-gggctcgagctagggaaatggggctcgcagg-3' (SEQ ID NO. 2)

To express the resulting PCR product, the PCR product was cloned into the animal cell expression vector pX0GC under the control of the CMV promoter. The pX0GC vector is a DHFR promoter that includes one or more CCGCCC repeats, and the DHFR-encoding nucleotide sequence is operably linked thereto (Korean Patent No. 880509). Specifically, the PCR product was digested with the restriction enzymes BamHI and XhoI at 37 ° C for 2 hours, and applied to a PCR purification kit (Qiagen, USA) to obtain a clipped DNA fragment. This DNA fragment was mixed with the pX0GC vector treated with the restriction enzymes BamHI and XhoI, and cloned using T4 DNA ligase to prepare a expression vector including the FacVII gene.

Example 2: Preparation of Expression Vectors for Expression of Various Recombinant FacVII Derivatives

The expression vector (pX0GC-FVII) comprising the FacVII gene prepared in Example 1 was used to obtain a nucleotide encoding a FacVII derivative having a portion of superoxide dismutase 1 (SOD1) at the C-terminus of FacVII. Sequence (SEQ ID NO. 30), and a performance vector capable of expressing the derivative.

Example 2-1: Preparation of recombinant FacVII derivative expression vector pX0GC-FVII-ATKAVC

A recombinant FacVII derivative expressing vector comprising a polynucleotide pX0GC-FVII-ATKAVC is further produced, the polynucleotide further having the SOD1 sequence encoding the expression vector pX0GC-FVII prepared in Example 1 at the 3' end of the FacVII gene Polynucleotides from the 1 to 6 sequence. In detail, using the expression vector pX0GC-FVII as a template, the following forward and reverse primers of SEQ ID NOS. 6 and 7 were used for PCR (95 ° C for 1 minute denaturation; 30 cycles (95 ° C for 60 seconds, 60 ° C 60 seconds and 68 ° C 90 seconds); 68 ° C 5 minutes). At this time, in order to facilitate the transformation, the recognition region of the restriction enzyme EcoRI was inserted into the primer of SEQ ID NO. 6, and the recognition region in which the restriction enzyme XhoI was inserted in the primer of SEQ ID NO. Subsequently, the nucleotide sequence of the approximately 1.4 kb PCR product obtained by PCR (SEQ ID NO. 8) was examined.

FVIIEcoRISS F (SEQ ID NO. 6): 5'-ccggaattcatggccaacgcgttcctggaggagctgcggccgggc-3'

FVII#1XhoIAS R(SEQ ID NO.7): 5'-ccgctcgagtcagcacacggccttcgtcgggggaaatggggctcgcaggaggactcctgggc-3'

To express the resulting PCR product, the PCR product was cloned into the animal cell expression vector pX0GC under the control of the CMV promoter. Specifically, with this limit The PCR products were digested with the enzymes EcoRI and XhoI for 2 hours at 37 ° C and applied to a PCR purification kit to obtain a sheared DNA fragment which was mixed with the restriction enzyme EcoRI and XhoI-treated pX0GC vector. And using T4 DNA ligase for selection, thereby preparing a expression vector (pX0GC-FVII-ATKAVC) having a polynucleotide encoding a FacVII derivative, which comprises a sequence from 1 to 6 encoding the SOD1 sequence linked to the 3' end of the FacVII gene. The polynucleotide of the sequence ATKAVC (SEQ ID NO. 5).

Example 2-2: Preparation of recombinant FacVII derivative expression vector pX0GC-FVII-GGGGSC

A recombinant FacVII derivative expressing vector comprising a polynucleotide, pX0GC-FVII-GGGGSC, further comprising 6 amines encoding the expression vector pX0GC-FVII prepared in Example 1 at the 3' end of the FacVII gene Polynucleotide of the base acid (GGGGSC, SEQ ID NO. 10). To complete this preparation, PCR was carried out in the same manner as in Example 2-1 except that the forward and reverse primers of SEQ ID NOS. 6 and 11 were encoded, and the nucleotide sequence of the PCR product of about 1.4 Kb was examined. (SEQ ID NOs. 12). Subsequently, the expression vector (pX0GC-FVII-GGGGSC) was prepared in the same manner as in Example 2-1 except that the coding product was used, and the vector contained the coding GGGGSC (SEQ ID NO. 10) at the 3' end of the FacVII gene. a polynucleotide encoding a FacVII derivative of a polynucleotide.

FVII#2XhoIAS R(SEQ ID NO.11): 5'-ccgctcgagtcagcaggagccgccgccgccgggaaatggggctcgcaggaggactcctgggc-3'

Example 2-3: Preparation of recombinant FacVII derivative expression vector pX0GC-FVII-SOD1 1-149

A recombinant FacVII derivative expression vector pX0GC-FVII-SOD1 1-149 comprising a polynucleotide (SEQ ID NO. 15) was further prepared, the polynucleotide further having the expression vector pX0GC-FVII included in Example 1 A polynucleotide of 1 to 149 amino acid of SOD1 (superoxide dismutase 1) at the 3' end of the FacVII gene.

In detail, the FacVII DNA sequence (SEQ ID NO. Minute denaturation; 30 cycles (95 ° C for 60 seconds, 60 ° C for 60 seconds, and 68 ° C for 90 seconds); 68 ° C for 5 minutes). At this time, in order to facilitate colonization, the recognition region of the restriction enzyme EcoRI was inserted into the primer of SEQ ID NO. 16, and the partial sequence at the 5' end of SOD1 was included in the primer of SEQ ID NO. As a result, the first PCR fragment was obtained.

FVIIEcoRISS F: 5'-ccggaattcatggtctcccaggccctcaggctcc-3' (SEQ ID NO. 16)

F VII SODInfAS R: 5'-cggccttcgtcgcgggaaatggggctcgcaggag-3' (SEQ ID NO. 17)

Next, the SOD1 cDNA (RC200725, OriGene, USA) was used as a template, and the SOD1 forward primer (SEQ ID NO. 18) and the SOD1 reverse primer (SEQ ID NO. 19) were used for PCR (95 ° C for 1 minute denaturation; 30 cycles (95 ° C 60 seconds, 60 ° C 60 seconds and 68 ° C 40 seconds); 68 ° C 5 minutes). At this time, in order to facilitate colonization, a partial sequence of the 3' end of FacVII is contained in the primer of SEQ ID NO. 18, and the recognition region of the restriction enzyme XhoI is inserted into the primer of SEQ ID NO. As a result, a second PCR fragment was obtained.

FVIISODInfSS F: 5'-gagccccatttcccgcgacgaaggccgtgtgcgt-3' (SEQ ID NO.18)

SODXhoIAS R: 5'-ccgctcgagtcaaattacaccacaagccaaacga-3' (SEQ ID NO. 19)

Thus, the obtained first and second PCR fragments were used as templates, and the FacVII forward primer (SEQ ID NO. 16) and the SOD1 reverse primer (SEQ ID NO. 19) were used for the second PCR. Finally, a third PCR fragment was obtained (PCR conditions: denaturation at 95 ° C for 1 minute; 30 cycles (95 ° C for 60 seconds, 60 ° C for 60 seconds, and 68 ° C for 120 seconds); 68 ° C for 5 minutes).

To express the resulting third PCR product, the PCR product was cloned into the animal cell expression vector pX0GC under the control of the CMV promoter. Specifically, the third PCR product was digested with the restriction enzymes EcoRI and XhoI at 37 ° C for 2 hours, and applied to a PCR purification kit to obtain a cleaved DNA fragment, the DNA fragment and the restriction enzyme. EcoRI and XhoI-treated pX0GC vectors were mixed and cloned using T4 DNA ligase to prepare a expression vector (pX0GC-FVII-SOD1 1-149) having a polynucleotide encoding a FacVII derivative, which comprises a linker to the FacVII gene 3 A polynucleotide encoding an amino acid from the sequence 1 to 149 of the SOD1 sequence.

Example 2-4: Preparation of recombinant FacVII derivative expression vector pX0GC-FVII-SOD1 IPRI

It is intended to insert the FacVII self-shearing point into the SOD1 gene included in the expression vector pX0GC-FVII-SOD1 1-149 prepared in Example 2-3. To accomplish this preparation, a recombinant FacVII derivative comprising the polynucleotide (SEQ ID NO. 21) was expressed as a vector pX0GC-FVII-SOD1 IPRI encoding an amino group of 1 to 149 by linking the mutated SOD1. Amino acid prepared by acid to the C-terminus of FacVII Sequence (SEQ ID NO. 20) by substitution of 7 to 10 amino acids (VLKG) of SOD1 in 451-454 amino acid of FacVII-SOD1 1-149 by IPRI.

In detail, PCR was carried out using the third PCR fragment obtained in Example 2-3 as a template and using a forward primer and a reverse primer (SEQ ID NOs. 22 and 23). Finally, a fourth PCR fragment was obtained (PCR conditions: 95 ° C 30 sec denaturation; 18 cycles (95 ° C 30 sec, 55 ° C 60 sec and 68 ° C 9 min); 68 ° C 9 min).

The nucleotide sequence of the PCR product of approximately 9 Kb was examined (SEQ ID NO. 21).

VIISOD1mutSS F: (SEQ ID NO. 22)

5'-cccgcgacgaaggccgtgtgcattccgaggatcgacggcccagtgcagggcatc-3'

FVIISOD1mutAS R: (SEQ ID NO. 23)

5'-gatgccctgcactgggccgtcgatcctcggaatgcacacggccttcgtcgcggg-3'

Subsequently, the fourth PCR product was digested with restriction enzyme DpnI at 37 ° C for 1 hour to cleave the non-mutated sequence, and was transformed into E. coli by transformation to prepare a expression vector (pX0GC-FVII-SOD1 IPRI). (The expression vector comprises a polynucleotide encoding a FacVII derivative of 1-149 amino acid having a mutated SOD1 at the C-terminus of FacVII).

Example 2-5: Preparation of Recombinant FacVII Derivative Expression Vector pX0GC-FVII-SOD1 1-25 IPRI

Preparation of a recombinant FacVII derivative comprising a polynucleotide (SEQ ID NO. 25) expression vector pX0GC-FVII-SOD1 1-25 IPRI encoding a 1 to 25 amino acid by linking the mutated SOD1 to The amino acid sequence (SEQ ID NO. 24) prepared at the C-terminus of FacVII was replaced by 7 to 10 amino acids (VLKG) of SOD1 by IPRI.

Specifically, the expression vector pX0GC-F VII-SOD1 IPRI prepared in Example 2-4 was used as a template, and PCR was carried out using a forward primer and a reverse primer (SEQ ID NOs. 16 and 26) (PCR conditions: 95 ° C Denaturation for 1 minute; 30 cycles (95 ° C for 60 seconds, 60 ° C for 60 seconds, and 68 ° C for 90 seconds); 68 ° C for 5 minutes). Finally, a fifth PCR fragment was obtained.

SOD1-25XhoIAS R: 5'-ccgctcgagtcaactttccttctgctcgaaattg-3' (SEQ ID NO. 26)

Subsequently, the fifth PCR product was digested with restriction enzymes EcoRI and XhoI at 37 ° C for 2 hours and applied to a PCR purification kit to obtain a cleaved DNA fragment, which was subjected to restriction enzymes EcoRI and XhoI. The treated pX0GC vector was mixed and cloned using T4 DNA ligase to prepare a expression vector (pX0GC-FVII-SOD1 1-25 IPRI) comprising a sequence encoding the mutated SOD1 linked to the C-terminus of the FacVII gene. A polynucleotide of a FacVII derivative of 1 to 25 amino acids.

Example 2-6: Preparation of Recombinant FacVII Derivative Expression Vector pX0GC-FVII-SOD1 1-90 IPRI

Preparation of a recombinant FacVII derivative comprising a polynucleotide (SEQ ID NO. 28) expression vector pX0GC-FVII-SOD1 1-90 IPRI encoding a 1 to 90 amino acid by linking the mutated SOD1 to The amino acid sequence (SEQ ID NO. 27) prepared at the C-terminus of FacVII was mutated by substituting IPRI for 7 to 10 amino acids (VLKG) of SOD1.

Specifically, the expression vector pX0GC-FVII-SOD1 IPRI prepared in Example 2-4 was used as a template, and PCR was carried out using a forward primer and a reverse primer (SEQ ID NOs. 16 and 29) (PCR conditions: 95 ° C 1 Minute denaturation; 30 cycles (95 ° C 60 Seconds, 60 ° C 60 seconds and 68 ° C 100 seconds); 68 ° C 5 minutes). Finally, a fifth PCR fragment was obtained.

SOD1-90XhoIAS R: 5'-ccgctcgagtcagtcagcagtcacattgcccaag-3' (SEQ ID NO. 29)

Subsequently, the fifth PCR product was digested with restriction enzymes EcoRI and XhoI at 37 ° C for 2 hours and applied to a PCR purification kit to obtain a cleaved DNA fragment, which was subjected to restriction enzymes EcoRI and XhoI. The treated pX0GC vector was mixed and cloned using T4 DNA ligase to prepare a expression vector (pX0GC-FVII-SOD1 1-90 IPRI) comprising a sequence encoding the mutated SOD1 linked to the C-terminus of the FacVII gene. A polynucleotide of a FacVII derivative of 1 to 90 amino acids.

Example 3: Performance of FacVII derivatives

Each of the expression carriers prepared in Example 2 was used to express various FacVII derivatives.

Example 3-1: Expression of FacVII derivatives in 293F cell lines

A expression vector (control group) and a expression vector (pX0GC-FVII-SOD1 1-149, pX0GC-FVII-SOD1 1-25 IPRI, pX0GC-F VII-SOD1 1-90 IPRI, which has nothing at the C-terminus of FacVII, pX0GC-F VII-ATKAVC or pX0GC-FVII-GGGGSC, each expression vector having a SOD1 1-149 sequence fused at the C-terminus of FacVII, a mutated SOD1 1-25 sequence, a mutated SOD1 1-90 sequence, SOD1 1-6 sequence, or prepared by the nucleotide sequence GGGGSC) introduced into the Freestyle ^TM 293 F cell line (Invitrogen, cat.no.R79007) Transformation of each strain to prepare different derivative-based FacVII for the Transformation of the strain which performed.

To this end, the 293F cell line was cultured every other day, and cultured at 37 ° C, 8% CO ₂ with shaking at 120 rpm or higher. When the number of cultured cells reached 10 x 10 ⁵ cells/ml and the survival rate was 85% or higher, each of the expression vectors prepared in Example 2 was introduced thereinto to obtain a transformant. In detail, 500μl of the maximum Freestyle ^TM reagent, 500 ug each of the expression vector and 10 ml of OptiproTM SFM (Invitrogen, cat.no.12309-050) was added 500 ml expressive cells / ml of medium in 10 x 10 ^5, and mixed They were placed at room temperature for 10 minutes to introduce each expression vector into the 293F cell line. Thereafter, 50 μg of vitamin K necessary for activation of FacVII was added thereto, and cultured at 37 ° C, 8% CO ₂ , shaking at 120 rpm or higher for 3 days. After the completion of the culture, the cells were centrifuged at 3000 rpm for 5 minutes to obtain a supernatant. Each of the FacVII derivatives incorporated in the supernatant was collected and purified. Subsequently, Western blotting was carried out using an anti-FacVII antibody to detect each of the expressed FacVII derivatives (Figs. 1a to 1c).

Figure 1a is a photograph showing the results of Western blot analysis of FacVII-ATKAVC in a 293F cell line, where M is a size marker (pre-stained protein marker, fermentas), and band 1 is FacVII-ATKAVC in a reduced state. According to the results of Western blot analysis, band 2 is the result of Western blot analysis of FacVII-ATKAVC in the non-reduced state. As shown in Figure 1a, about 20 to 30% of the dimer pattern was detected in the non-reduced state, and no dimer pattern was detected in the reduced state.

Further, Fig. 1b is a photograph showing the results of Western blot analysis of the control group and FacVII-GGGGSC in the 293F cell line, and Fig. 1b shows the appearance of FacVII-ATKAVC and FacVII-SOD1 1-149 in the 293F cell line. A photograph of the results of Western blot analysis of molecular weight differences.

As shown in Figures 1b and 1c, various FacVII derivatives were found to be normally produced in 293F cell lines.

Example 3-2: Expression of a FacVII derivative in a CHO cell line

The expression vector prepared in Example 2-1 was introduced into a DG44/CHO cell line (CHO/dhfr-) (this cell line lacks DHFR to show incomplete DNA synthesis) (Urlaub et al., Somat. Cell. Mol. Genet., 12, 555-566, 1986)) to obtain a transformant strain from which the FaVII-ATKAVC derivative was expressed.

Further, the DG44/CHO cell strain was cultured to achieve 80% to 90% confluence, and the cells were washed 3 times with Opti-MEM (Gibco, cat. No. 51985034).

On the other hand, a mixture of 3 ml of Opti-MEM and 5 μg of the expression vector pX0GC-FVII-ATKAVC, and 3 ml of Opti-MEM and 20 μl of lipofectamine (Gibco, cat. no. 18324-012) were each placed at room temperature for 30 minutes. Subsequently, the mixture was mixed and added to the cultured DG44/CHO cell line. Then, the cells were cultured at 37 ° C, 5% CO _{2 for} about 18 hours, and the expression vector pX0GC-FVII-ATKAVC was introduced into the DG44/CHO cell line. Subsequently, the cultured cells were washed three times with DMEM-F12 (Gibco, cat. no. 11330) supplemented with 10% FBS, and then the medium was added thereto, followed by culture for 48 hours. The cultured cells were detached by trypsin treatment and inoculated into MEM-α medium containing 10% FBS and 1 mg/ml of G418 (Cellgro, cat. no. 61-234-RG) (WELGENE , cat.no.LM008-02)), the medium has no selection medium (HT supplement (hexahydroxyindole-thymidine)). Until the transformed cells survived to form a colony, the medium was replaced with selective medium every 2 or 3 days. Thus, the transformed cell line is selected from the group of isolated cells. At this time, in order to increase the expression level of the FacVII-ATKAVC derivative in the selected transformed cells, 10 nM MTX (Sigma, cat. no. M8407) was added to the selection medium to gradually increase the concentration, after 2 to 3 weeks, The MTX content was increased to 30 nM.

Furthermore, in order to reduce the heterogeneity of transduced cells, a limiting dilution method was performed to obtain a single colony. In detail, the transduced cells were diluted to a ratio of 0.7 fines per well in a 96-well plate, and cultured for 2 to 3 weeks to detect whether a single colony could be observed. When a cluster formation of a single colony was observed in the wells, the single colony was transferred to a 24-well plate, and the cell growth rate and the expression level of the FacVII derivative of each colony were analyzed by ELISA, and the expression of the highest FacVII derivative was selected. Horizontal community. Designated as "HMF709" and deposited in the Korea National Type Culture Collection, Korea Research Institute for Type Culture, Korean Research Institute of Bioscience and Biotechnology (111 Gwahangno, Yuseong-gu, Daejeon, Korea)), the registration number is "KCTC12022BP" (domestic registration number BCRC 960455).

Example 4: Purification of FacVII-ATKAVC

The transformant strain prepared in Example 3-2 was cultured to express FacVII-ATKAVC, and the culture solution was centrifuged at 3000 rpm for 5 minutes to obtain a supernatant.

The supernatant was filtered using a 0.2 μm microfiltration membrane, and 0.6 M ammonium sulfate was added thereto, and the mixture was applied to a butyl HP column using a concentration gradient buffer solution (20 mM Tris-) containing 0.6 to 0 M ammonium sulfate. The HCl pH 7.5) was stripped to obtain an active fraction comprising FacVII-ATKAVC.

The resulting active portion of the buffer solution was replaced with a 10 mM sodium phosphate buffer solution (pH 7.0), applied to a Heparin HP column, and washed with a 0 to 1.0 M NaCl concentration gradient buffer solution (10 mM sodium phosphate, pH 7.0). For use, to obtain an active moiety comprising FacVII-ATKAVC.

The active fraction was concentrated and applied to a Superdex 75 column and then flushed with a 150 mM NaCl 20 mM Tris-HCl (pH 7.5) buffer solution to obtain an active fraction comprising FacVII-ATKAVC. The buffer solution of the obtained active fraction was replaced with a 2 mM benzamidine 20 mM Tris-HCl (pH 7.5) buffer solution, which was applied to a Q FF column. Then, washing (2 mM benzamidine 0.2 M NaCl in 20 mM Tris-HCl (pH 8.0) buffer) and re-equilibration (2 mM benzamidine 0.1 M NaCl 20 mM Tris-HCl (pH 8.0) buffer) The FacVII-ATKAVC was purified by concentration gradient elution (2 mM benzamidine 25 mM NaCl 35 mM CaCl ₂ , 20 mM Tris-HCl (pH 8.0) buffer).

The purity of the purified FacVII-ATKAVC was examined by SDS PAGE (Fig. 2). Fig. 2 is a photograph showing the results of electrophoresis of purified FacVII-ATKAVC, wherein M is a size marker, strip 1 is a reduced state of FacVII, strip 2 is a reduced state of FacVII-ATKAVC, and strip 3 is a non-reduced state. In the case of FacVII, the band 4 is a non-reducing state of FacVII-ATKAVC.

Example 5: Preparation of a conjugate of FacVII-ATKAVC and PEG

The FacVII-ATKAVC purified in Example 4 was bonded to PEG having a different molecular weight to prepare a conjugate.

Example 5-1: Preparation of FacVII-ATKAVC-40 kDa PEG conjugate

In order to PEGylate the C-terminus of FacVII-ATKAVC with 40 kDa mPEG-maleimide (NOF, Japan), in the presence of 100 mM phosphate buffer solution (pH 5.5) and a reducing agent, a molar of 1:20 2 mM of triarylphosphine was added thereto by mixing FacVII-ATKAVC (1 mg/ml) and 40 kDa mPEG-maleimine, and reacted at 25 ° C for 2 hours. As a result, monoPEGylated FacVII-ATKAVC (FacVII-ATKAVC-40k PEG conjugate) was prepared (Fig. 3). Figure 3 is a photograph showing the results of electrophoresis of a conjugate of FacVII-ATKAVC and PEG, wherein M is a size marker, a strip 1 is a non-reducing FacVII-ATKAVC-40 kDa PEG conjugate, and band 2 is a non-reducing FacVII-ATKAVC-5 kDa PEG conjugate.

Example 5-2: Preparation of FacVII-ATKAVC-5 kDa PEG Conjugate

Acetaldehyde-5 kDa PEG-Malay was carried out in the same manner as in Example 5-1 except that acetaldehyde-5 kDa PEG-maleimide was used instead of 40 kDa mPEG-maleimide. The quinone imine PEGylates the C-terminus of FacVII-ATKAVC to prepare a PEGylated FacVII-ATKAVC (FacVII-ATKAVC-5 kDa PEG conjugate) (Fig. 3).

Example 5-3: Preparation of FacVII-ATKAVC-PEG-Fc Complex

In order to link the acetaldehyde reactive group of maleimine-10 kDa PEG-acetaldehyde (NOF, Japan) to the N-terminus of the immunoglobulin Fc region, the presence of a 100 mM phosphate buffer solution (pH 6.0) and a reducing agent The immunoglobulin Fc region and maleic imine-10 kDa PEG-acetaldehyde were mixed at a molar ratio of 1:1, and 20 mM Na-CNBH _{3 was} obtained at a protein concentration of 10 mg/ml. Join it. The mixture was reacted at low temperature (4 to 8 ° C) for 2 hours. To obtain a monoPEGylated immunoglobulin Fc region (maleimide-10 kDa PEG-Fc), use Source 15Q for cation exchange chromatography and a sodium chloride concentration gradient in 20 mM Tris buffer (pH) 7.5) Carry out the flushing.

On the other hand, the reducing agent and 0.5 to 2 mM triphenylphosphine-3,3',3'-trisulphonic acid trisodium salt hydrate were used to reduce FacVII at room temperature in a 10 mM diglycine peptide buffer solution (pH 5.5). - The C-side of ATKAVC is 2 hours.

The C-terminally reduced FacVII-ATKAVC and the monoPEGylated immunoglobulin Fc region (maleimide-10kDa PEG-Fc) were mixed in a molar ratio of 1:4 to 1:20 in a 50 mM Tris buffer solution. (pH 7.5) and total protein concentration It was reacted at room temperature for 2 hours at 1 to 2 mg/ml. First, cation exchange chromatography was performed using Source 15Q, and the FacVII-ATKAVC-10k PEG-Fc complex was eluted using a sodium chloride concentration gradient in a 20 mM Tris buffer solution (pH 7.5).

In order to activate FacVII in the FacVII-ATKAVC-10k PEG-Fc complex, solution reaction was carried out in a 0.1 M Tris-HCL buffer solution (pH 8.0) at about 4 mg/ml on a FacVII basis and at a low temperature (4 to 8 ° C). 18 hours.

Particle size exclusion chromatography was performed using Superdex 200 and 10 mM glycil-glycine buffer solution (pH 5.5) to purify the final FacVIIa-ATKAVC-PEG-Fc.

Purified FacVIIa-ATKAVC-PEG-Fc purity was determined by SDS PAGE and Western blotting (Fig. 4). Figure 4a is a photograph showing the results of electrophoresis of the purified FacVIIa-ATKAVC-PEG-Fc conjugate, wherein M is a size marker, band 1 is a reduced state of FacVIIa-ATKAVC-PEG-Fc, and band 2 is a non- Reduced state of FacVIIa-ATKAVC-PEG-Fc. Figure 4b is a photograph showing the results of Western blot analysis of purified FacVIIa-ATKAVC-PEG-Fc, wherein band 1 is a reduced state of FacVIIa-ATKAVC-PEG-Fc, and band 2 is a non-reducing form of FacVIIa- ATKAVC-PEG-Fc.

Example 6: In vitro activity (EC ₅₀ ) of FacVII and FacVII-ATKAVC

For the in vitro activity of FacVII and FacVII-ATKAVC, a commercially available kit was used for color development analysis, and activity analysis was performed according to the European Pharmacopoeia "2.7.10. ASSAY OF HUMAN COAGULATION FACTOR VII".

The diluted FacVII and FacVII-ATKAVC were activated by thromboplastin and Ca ²⁺ ions. FX is activated to FXa by activated FacVIIa and FacVIIa-ATKAVC, and the substrate S-2765 (Na-Cbo-D-Arg-Gly-Arg-pNA) is hydrolyzed by activated FXa and dissociated into peptides and hairs. Color group pNA. The absorbance of the dissociated pNA at 405 nm was monitored to determine the in vitro activity of FacVIIa and FacVIIa-ATKAVC.

Absorbance changes according to the concentrations of FacVII and FacVII-ATKAVC were detected by using a 4-parameter model of Softmax Pro 4.0 software, and the activity between the two substances was compared using the obtained EC ₅₀ values.

The test results (Fig. 5 and Table 1) show that the in vitro activity of FacVII-ATKAVC is equivalent to or higher than that seen by natural FacVII.

As shown in Tables 1 and 5, it was found that FacVII and FacVII-ATKAVC exhibited comparable in vitro activities, indicating that the FacVII and FacVII derivatives of the present invention have activity comparable to the native form, and the addition of the peptide linker to the C-terminus is not Will affect its activity.

Example 7: In vitro activity FacⅦ-ATKAVC and FacⅦ-ATKAVC-40 kDa PEG's (EC ₅₀₎

To determine the activity according to site-specific conjugation, the in vitro activities of FacVII-40 kDa PEG, FacVII-ATKAVC and C-terminally PEGylated FacVII-ATKAVC-40 kDa PEG were determined. Color development analysis was carried out using a commercially available kit (Chromogenix, COASET), which was carried out in the same manner as in Example 6. The absorbance change according to the concentration of the test sample was detected using a 4-parameter model of Softmax Pro 4.0 software, and the relative activity after PEGylation was determined using the obtained EC ₅₀ value.

The results of this assay (Table 2) show that the in vitro activity of the N-terminally PEGylated FacVII-40 kDa PEG is about 11% higher than that of FacVII, and the in vitro active phase of the C-terminally PEGylated FacVII-ATKAVC-40 kDa PEG. It has a price of about 29% compared to FacVII-ATKAVC.

The conjugate activity of the PEG of the present invention with the FacVII derivative showed a valence of about 29% compared to the FacVII derivative. Conversely, the conjugate activity of the PEG with FacVII showed a valence of about 11% compared to FacVII. These results indicate that the C-terminus of the PEG FacVII-ATKAVC-40 kDa PEG of about 2.5 times to maintain a higher relative activity (EC ₅₀ as compared to the N-terminus of the PEG FacVII-40 kDa PEG's). And the FacVII activity can be highly maintained by using point-specific ligation of ATKAVC.

Example 8: In vitro activity of FacVIIa-ATKAVC-PEG-Fc

The in vitro activity of FacVIIa-ATKAVC-PEG-Fc was determined using a commercially available kit (Staclot Vlla-rTF, Stago) and the international standard NIBSC Factor VIIa (656 IU/vial, Code No. 07/228) as a standard substance. It is based on coagulation by a specific reaction of rsTF (recombinant soluble tissue factor) and Factor VIIa. NIBSC Factor VIIa, FacVIIa-ATKAVC, and FacVIIa-ATKAVC-PEG-Fc were diluted in a 1:1 ratio in the absence of FacVII human serum and reacted with a mixture of rsTF and phospholipids for approximately 180 seconds. Thereafter, 25 mM CaCl ₂ was added thereto to measure the clotting time. As the content of Factor VIIa increases, the clotting time changes briefly.

To calculate the specific activity (IU/mg) of FacVIIa-ATKAVC and FacVIIa-ATKAVC-PEG-Fc, the first time using PLA 2.0 analysis, FacVIIa-ATKAVC and FacVIIa-ATKAVC- relative to the titer (IU/mL) of NIBSC Factor VIIa The titer of PEG-Fc (IU/mL). Thereafter, the calculated potency (IU/mL) was divided by the protein concentration (mg/mL) to calculate the specific activity.

The results of this assay (Table 3) show that the in vitro activity of FacVIIa-ATKAVC-PEG-Fc is approximately 20632 IU/mg, indicating that the FacVIIa-ATKAVC-PEG-Fc maintains approximately 45% activity compared to FacVIIa-ATKAVC.

【Biomaterial Storage】

Foreign deposit information [please note according to the country, organization, date, number order]

Korea, Korea Typical Culture Collection Center, September 23, 2011, KCTC 12022BP

<110> HANMI SCIENCE CO., LTD.

<120> Derivatives of clotting factors VII and VIIa, conjugates, and composites containing the same and uses thereof (BLOOD COAGULATION FACTOR VII AND VIIa DERIVATIVES, CONJUGATES AND COMPLEXES COMPRISING THE SAME, AND USE THEREOF)

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<151> 2011-10-06

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The representative figure has no component symbols and the meaning of its representation.

Claims (44)

A derivative of Factor VII (FacVII) or its active Factor VIIa (FacVIIa) comprising an amino acid sequence of Factor VII (Factor VII, FacVII) and a linker at its C-terminus A peptide linker, wherein the peptide linker is a partial sequence of superoxide dismutase 1 . The derivative of claim 1, wherein the C-terminal amino acid residue of the peptide linker is cysteine. The derivative according to claim 1, wherein the partial sequence of the superoxide dismutase 1 is by a self-shearing point sequence IPRI (isoleucine-proline-arginine-iso The leucine acid is mutated by substituting VLKG (proline- lysine-lysine-glycine) in the sequence. The derivative according to claim 1, wherein the derivative is composed of an amino acid sequence selected from the group consisting of SEQ ID NOS. 9, 13, 14, 20, 24 and 27. A polynucleotide encoding a derivative of FacVII and its active form of FacVIIa as described in any one of claims 1 to 4. The polynucleotide of claim 5, wherein the polynucleotide is composed of a nucleotide sequence selected from the group consisting of SEQ ID NOs. 8, 12, 15, 21, 25 and 28. . A performance vector comprising the polynucleotide of claim 5 of the patent application. The expression vector of claim 7, wherein the expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos. 8, 12, 15, 21, 25 and 28. A transformant strain comprising the expression vector of claim 7 of the patent application. The transgenic strain according to claim 9, wherein the transgenic strain has a registration number of BCRC 960455. A method for producing a FacVII derivative, comprising the steps of: (i) cultivating a transformant according to claim 9 for obtaining a culture solution; and (ii) recovering the FacVII derivative from the culture solution. The method of claim 11, wherein the step of activating the recovered FacVII derivative is further included. A conjugate of FacVII or an active form thereof, wherein the non-peptidyl polymer capable of prolonging the half-life of blood is linked to the derivative of FacVII or its active form of FacVIIa as described in any one of claims 1 to 4. The peptide linker of the substance. The conjugate of claim 13, wherein the non-peptidyl polymerized product is linked to the C-terminus of the derivative of the FacVII or its active form FacVIIa. The conjugate of claim 13, wherein the non-peptidyl polymer has one or more selected from the group consisting of acetaldehyde, propionaldehyde, butyraldehyde, maleimine, or pyridyl disulfide a reactive group of a group consisting of a thiol and an amber quinone derivative. The conjugate of claim 15, wherein the succinimide derivative is amber succinimide propionate, carboxymethyl amber succinimide, hydroxy amber succinimide or Amber succinimide carbonate. The conjugate of claim 13, wherein the non-peptidyl polymer has two or three reactive ends. The conjugate of claim 13, wherein the non-peptidyl polymerization The substance has a maleic imine reactive group or an acetaldehyde reactive group at both ends. The conjugate of claim 13, wherein the non-peptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol non-peptidyl copolymer, polyoxyethylated polyol, A group consisting of polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid non-peptidyl polymers, chitin, hyaluronic acid, and combinations thereof. The conjugate of claim 13, wherein one end of the non-peptidyl polymer constituting the FacVII conjugate or the FacVIIa conjugate is linked to a group selected from the group consisting of an antibody, an albumin, and a transferrin. Carrier. A complex of FacVII or an active form thereof, which is linked to a immunoglobulin via a non-peptidyl polymer by a derivative of FacVII or FacVIIa as described in any one of claims 1 to 4 The protein Fc region is composed. The composite according to claim 21, wherein the non-peptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol. a group consisting of polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, and combinations thereof. The composite of claim 21, wherein the non-peptidyl polymer has one, two or three reactive ends. The composite according to claim 21, wherein the non-peptidyl polymer has a maleic imine reactive group or an acetaldehyde reactive group at each end. The complex of claim 21, wherein the immunoglobulin Fc region is composed of one to four domains selected from the group consisting of CH1, CH2, CH3 and CH4 domains. The complex of claim 21, wherein the immunoglobulin Fc region comprises a hinge region. The complex of claim 21, wherein the immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE or IgM. The complex of claim 21, wherein the immunoglobulin Fc region is an IgG4 Fc region. The complex of claim 21, wherein the immunoglobulin Fc region is a human aglycosylated IgG4 Fc region. A method of preparing a FacVIIa complex comprising the steps of: (i) covalently linking a non-peptidyl polymer having an amber quinone imine derivative or an acetaldehyde reactive group at one end to an immunoglobulin at pH 5.0 to pH 9.0 An amine group of the protein Fc; (ii) recovering a conjugate comprising the immunoglobulin Fc region covalently linked to the amine group and the non-peptidyl polymer from the reaction mixture of the step (i); (iii) covalent linkage as in the application The C-terminal thiol group of the derivative of FacVII according to any one of claims 1 to 4, to the other end of the non-peptidyl polymer of the recovered conjugate, for producing the non-peptidyl group Each of the ends of the polymer has the immunoglobulin Fc region and a FacVII complex of the FacVIIa derivative; and (iv) activating the FacVII complex produced in step (iii) for producing a linkage via the non-peptidyl polymer FacVIIa and the FacVIIa complex of the immunoglobulin Fc region. A method of preparing a FacVIIa complex comprising the steps of: (i) covalently linking at one end to an amber quinone imine derivative at pH 5.0 to pH 9.0 Or a non-peptidyl polymer of an acetaldehyde-reactive group to an amine group of an immunoglobulin Fc; (ii) recovering from the reaction mixture of step (i) comprising the immunoglobulin covalently linked with an amine group and a non-peptidyl polymer a conjugate of the protein Fc region; (iii) covalently linking the C-terminal thiol group of the FacVIIa derivative according to any one of claims 1 to 4 to the non-peptide of the recovered conjugate The other end of the base polymer is used to produce a FacVIIa complex having the immunoglobulin Fc region and the FacVIIa derivative at each end of the non-peptidyl polymer. A method of preparing a FacVIIa conjugate comprising the steps of: (i) covalently bonding selected from the group consisting of acetaldehyde, propionaldehyde, butyraldehyde, maleimine, ortho-pyridyl disulfide, thiol, and amber The non-peptidyl polymer of the reactive group of the group consisting of the quinone imine derivatives to the C-terminal thiol group of the FacVII derivative according to any one of claims 1 to 4; Ii) recovering the FacVII conjugate prepared by covalently linking the non-peptidyl polymer to the FacVII derivative; (iii) activating the recovered FacVII conjugate to produce the non-peptidyl group having a link to the FacVIIa region Polymeric FacVIIa conjugate. A method of preparing a FacVIIa conjugate comprising the steps of: (1) covalently bonding having an aldehyde group selected from the group consisting of acetaldehyde, propionaldehyde, butyraldehyde, maleimine, ortho-pyridyl disulfide, thiol, and amber The non-peptidyl polymer of the reactive group of the group consisting of the quinone imine derivatives to the C-terminal thiol group of the FacVIIa derivative as described in any one of claims 1 to 4; as well as (ii) recovering the FacVIIa conjugate prepared by covalently linking the non-peptidyl polymer to the FacVIIa derivative. A method for preparing a FacVIIa complex, which is linked to the other end of the non-peptidyl polymer by an immunoglobulin Fc region linked to a recovered FacVIIa conjugate as described in claim 32 or claim 33 . The method of claim 30, wherein the activation of the FacVII derivative, the FacVII conjugate or the FacVII complex is carried out via activation in a column or activation in solution. The method of any one of claims 30 to 33, wherein the non-peptidyl polymer is polyethylene glycol. A complex of FacVII or an active form of FacVIIa, which is obtained from a FacVII derivative according to any one of claims 1 to 4 or a derivative of the FacVIIa via a polyethylene glycol, Polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin The non-peptidyl polymer of the group consisting of hyaluronic acid and a combination thereof is linked with a carrier. The complex of claim 37, wherein the carrier is selected from the group consisting of an antibody, albumin and transferrin. The composite of claim 37, wherein the non-peptidyl polymer has one, two or three reactive ends. The composite according to claim 37, wherein the non-peptidyl polymer has a maleic imine reactive group or an acetaldehyde reactive group at each end. A pharmaceutical composition for preventing or treating hemophilia, which comprises a FacVII derivative or a FacVIIa derivative as described in any one of claims 1 to 4. The FacVII conjugate or the FacVIIa conjugate according to any one of claims 13 to 20, or the FacVII complex according to any one of claims 21 to 29 Or the FacVIIa complex as an active ingredient. A pharmaceutical composition for promoting blood clotting, which comprises a FacVII derivative or a FacVIIa derivative according to any one of claims 1 to 4, as in any one of claims 13 to 20. The FacVII conjugate or the FacVIIa conjugate, or the FacVII complex or the FacVIIa complex as described in any one of claims 21 to 29, as an active ingredient. A therapeutically effective amount of a pharmaceutical composition according to claim 41, for use in the manufacture of a medicament for preventing or treating hemophilia in an individual. A therapeutically effective amount of a pharmaceutical composition according to claim 42 for use in the manufacture of a medicament for promoting coagulation in an individual.